Increased Expression Of HERV-K Related Novel MRNA In Head And Neck Cancer Tissues

BackgroundHuman endogenous retrovirus (HERV) originated from integration of retrovirus into the human genome millions of years ago through the germ line infection, have been documented to play various functional roles in the human genome through various modes such as retrotransposition, insertional mutagenesis and proto-oncogen activation. Most of the promoters and enhancers that play a pivotal role in human gene regulation lie in the long terminal region (LTR region) of HERV. Among all sub families of HERV, HERV K is found to be most potent one with active open reading frame (ORF) of env and gag protein. The two important oncoproteins Rec and Np9, which are spliced products of HERV-K env gene, were detected in melanomas, breast cancers and some other cancers, especially in germ cell tumors like teratocarcinoma.Materials and methodsReverse transcription polymerase chain reaction (RT-PCR), both semiquantitative and quantitative methods, later one using SYBR green real master mix, were done in different cell lines and cancer and non cancer patients’tissues from head and neck region. Total71tissue samples were processed for RNA extraction and two step RT-PCR. TA cloning and sequencing of around300bp PCR product with Rec primers were performed. The sequence results were analyzed using BLAST program and ORF Finder (Open Reading Frame Finder) from NCBI and also by signal4.0server to determine signal peptide within the amino acid sequence of ORF.ResultIn this study, we first chose laryngeal carcinoma cell line Hep2, human embryonic kidney cell line Hek293and cancer and non cancer head and neck tissues to detect the expression of HERV K rec gene using the primers once used by Buscher et al in their study of the HERV K rec gene expression in melanoma. In this primer pairs, splice donor and splice acceptor were located at its downstream and upstream respectively. Using this primer, we detected two splicing products of Env gene, one double spliced product containing rec sequence (about600bp) and another single spliced product without containing rec sequence (about300bp) in Hep2cells but only one double spliced product in Hek293cells. In case of both cancer and non cancer head and neck tissues, we observed very rare or undetectable number of double spliced product of Env gene whereas about50%of single spliced product of Env gene was detected in these tissues. This single spliced product of mRNA is having a total length of approximately1.5kb and its function is yet unknown. This non rec sequence was found to be having338bp when sequenced after doing TA cloning. Application of NCBI BLAST analysis reveals this single spliced non rec product of338bp sequence is highly homologous to part of HERV-K LTR sequences and also highly homologous to the gene sequences of unknown function from teratoma cells and embryonic stem sources. The338bp sequence contains54amino acids and found to consist of short length open reading frame with the presence of signal peptide as well. The downstream primer we used was found to be degenerate primer with multiple different base pairs, while taking into account the Genbank reports of HERV-K and this variation could affect the gene expression and its stability. Hence, considering this fact, we again designed a new primer pairs on the basis of338bp and referred it as P2primers. These P2primers was then subjected to real time PCR with cell lines and both cancer and non cancer head and neck tissue to get an amplification product of315bp. The final result showed high expression rate (80%) of315bp sequence in head and neck cancer tissues and very low expression rate (17.2%) in non cancer head and neck tissues showing a measurable difference between cancer and non cancer tissues (showed a significant chi square test; p<0.0001). In addition, there was positive expression of this315bp sequence in all four cell lines using P2primer. However, there showed no statistical significance when comparing its expression level.ConclusionA high differences between cancer and non-cancer head and neck tissues in the expression of novel HERV-K related315bp mRNA indicates that this new mRNA could have significant role in augmentation of cancer. Another, the homology of this novel sequence with part of HERV K LTR region and unknown mRNA sequences from teratocarcinoma and germ cells suggests that the sequence being related to HERV K may have a role in cell proliferation and or cell differentiation. In addition, a few findings showing the presence of ORF and a signal peptide within one ORF suggest that this novel mRNA could be translated into some specific protein regulating cell proliferation or else its DNA sequence could act as retrotransposon linking with other specific gene aiding in cell proliferation or in cancer pathogenesis, but due to lack of proper evidence and further experiment, its function could not be made clear. Hence, further detailed experiments and researches should be carried out to elaborate its specific function and significance in the human genome.